Many imaging modalities have been developed for non-invasive evaluation of heart disease. Radiotracers that can assess myocardial perfusion and heart metabolism are useful for clinical evaluation of ischemic heart disease and cardiomyopathies. Fatty acids (FAs) are the principal substrate for the production of adenosine triphosphate (ATP) in the myocardium under aerobic conditions. Nearly 70% of myocardial energy results from metabolism of fat in the basal state, with the remainder of the myocardial energy requirements supplied by glucose (15%), lactate and pyruvate (12%) and amino acids (5%). Therefore, FAs and modified fatty acids (MFAs) have been proposed for imaging the heart.
Uptake of free FAs by the myocardium occurs at an extraction percentage of 40% to 60% of blood content, which is proportional to perfusion. Transported to the heart as nonesterified FAs, as triglycerides in very low-density lipoprotein particles or in chylomicrons, or bound to serum albumin, they pass along concentration gradients to the interstitium. Under these conditions, FAs supply as much as 70% of oxidatively metabolized substrate. The extraction of free FAs by the myocyte is regulated by several variables including FA chain length, the availability of other metabolic substrates, circulating levels of hormones, cardiac workload, and the presence or absence of ischemia.
MFAs behave like native FAs up to the β-oxidation step in the mitochondria, where it is sequestered for a long period of time (Livni, E. et al. 1990 Lipids 25: 238-40). Highly simplified, the fate of an FA may be described as follows: FA passes from capillary blood into the interstitial space. It may “back-diffuse” to the vascular space or pass through the interstitial space, where it may become activated as acyl-coenzymeA (CoA). The activated FA can then be esterified to form triglycerides, incorporated into phospholipids or carried into the mitochondria and oxidized. The activation of FA to acyl-CoA requires energy and is believed to be essentially irreversible in vivo. Since acyl-CoA cannot escape through the cell membrane, it becomes trapped in the cell. However, the formation of triglycerides is not irreversible and these can be broken down into the constituent FA and glycerol, adding to the FA pool.
As described above, there is an intimate relationship between FA metabolism and myocardial integrity. As a result of their high rate of utilization, labeled FAs in conjunction with suitable detection techniques can provide a means of quantifying in vivo regional myocardial metabolism. Two approaches can be employed to quantify the utilization of substrates in vivo. The first involves the use of radiolabeled, positron emitting physiologic substrates. This approach has been used to assess glucose and FA metabolism in the heart with carbon 11 (11C) palmitate (PA) in rabbit (Raichle, M. E. et al. (1978) Science 199: 986-987). It has been demonstrated that after brief intervals of ischemia, PA extraction fell markedly, even when alterations in cardiac function were reversible (Klein M. S. et al (1979) Am. J. Physiol. 237: H51-H58; Goldstein R. A. et al (1980) J. Nucl. Med. 21: 342-348; Weiss, E. S. et al. (1969) Circulation 19: 25-32; Fox, K. A. et al. (1975) Circ. Res. 57: 232-243; Hoffman, E. J. et al. (1977) J. Nucl. Med. 18: 57-61; Ter-Pogossian, M. M. et al. (1980) Circulation 61: 242-255; Weiss E. S. et al. (1977) Circulation 55: 66-73; Schon, H. R. et al. (1982) Am. Heart J. 103: 532-547; Schon, H. R. et al. (1982) Am. Heart J. 103: 548-561; Schelbert, H. R. et al. (1983) Am. Heart J. 105: 492-504; Schelbert, H. R. et al (1983) Am. Heart J. 106: 736-50; Sobel, B. E. (1982) Am. Heart J. 103: 673-681; Schelbert, H. R. et al. (1983) Am. Heart J. 105: 522-526; Schelbert, H. R. (1985) Circulation 72: TV122-133; Sobel, B. E. (1985) Circulation 72:IV22-30; Rosamond, T. L. et al (1987) J. Nucl. Med. 28: 1322-1329; Grover-McKay, M. et al (1986) Circulation 74: 281-292; Schelbert, H. R. et al (1986) Am. Heart J. 111: 1055-1064; Jaffe, A. S. et al (1987) Int. J. Cardiol. 15: 77-89; Knabb, R. M. et al (1987) J. Nucl. Med. 28:1563-1570; Myars, D. W. et al (1987) Am. J. Physiol. 253: 107-114). In addition, zones of persistently decreased flow also showed decreased PA extraction (Schelbert, H. R. (1985) Circulation 72: TV122-133).
Other studies using 11C-PA provide the experimental basis for studying regional myocardial FA distribution. The application of physiologic radiolabeled FAs to in vivo quantification of regional myocardial metabolic rates suffers from several drawbacks. First, the use of 11C-FAs labeled with 11C on the carboxyl group is subject to loss of labeling during the first round of β-oxidation. Studies employing direct intracoronary administration of 11CO2 and direct myocardial monitoring demonstrate the evolution of 11CO2 within 30 seconds and a 50% clearance within 2-8 minutes. Secondly, the rapid washout of the radiolabel due to β-oxidation and short sequential imaging periods imposes limitations on counting statistics. Third, in the myocardial cell, FAs are distributed among different pools: free FAs, triglycerides, phospholipids, diglycerides, and monoglycerides. It is unclear whether the rate of FA oxidation is proportional to the rate of triglyceride hydrolysis. After the initial clearance of radioactivity from the blood, there is a rise in activity due to the release of radiolabeled metabolites from the liver. This makes the completion of labeled FA detection necessary before the rise occurs. Other studies in dogs using 11C-PA showed that during ischemia, quantitation is limited due to the complexity of its metabolic fate (Schelbert et al., 1983 Am. Heart J 106:736-50). 11C acetate has been proposed as an alternative due to its simpler metabolic fate.
The second approach involves the use of “analog tracers” that enter a known metabolic pathway. However, because of their unique chemical structure, metabolism of these tracers stop at a certain stage, leaving the radiolabel trapped in the cell in a known form. This concept has been applied to the study of glucose metabolism using glucose analogs such as 1-[11C]-2-deoxyglucose (2DG) and 2-[18F]-fluorodeoxyglucose (2FDG). The principle of metabolic trapping has been used successfully with 2FDG to measure in vivo regional glucose metabolic rates in humans. Investigations of the use of 2FDG for measuring myocardial glucose metabolism have been conducted. Similarly, FAs have also been widely used to measure metabolic activity in the myocardium. A major drawback to the use of FAs is the quick washout rates, as alluded above. FAs tend to wash out very quickly due to β-oxidation, depending on the position of the radionuclide. Subsequently, the radiolabeled FA or metabolites can then accumulate in tissues other than the region of interest, primarily liver and lung. In radiohalogenated aliphatic fatty acids, such accumulation occurs frequently with 123I, which migrates and is stored in the thyroid gland, and 18F, which is stored in bone.
Evans and coworkers radiolabeled straight-chain FAs by saturation of the double carbon bond of oleic acid with 131I and found that although photoscans of the canine heart were produced, the low specific activity of the final product precluded its clinical use (Evans, J. et al (1965) Circ. Res. 16: 1-10; reviewed in Corbett, J. R., (1999) Semin. Nucl. Med. 29(3): 1999-2006). Since then, there have been many MFAs developed for cardiac imaging. Poe and coworkers showed that [123I]-hexadecanoic acid (IHXA) and [123I]-heptadecanoic acid (IHDA) were indicators of myocardial perfusion in experimental canine models and demonstrated clearance rates similar to that of 11C-PA (Poe, N. et al, (1976) J. Nucl. Med. 16; 17-21; Poe, N. et al, (1977) Radiology 124: 419-424). All subjects with prior myocardial infarcts showed decreased regional tracer uptake. Machulla et al reported that the ω-terminal labeled FAs were more efficiently extracted than analogs labeled in the α-position, and that IHDA had the highest uptake (Machulla, H. et al (1978) J. Nucl. Med. 19: 298-302).
Although IHDA and IHXA have potential as myocardial perfusion agents, their ability to access myocardial metabolism in patients has been questioned (Freundlieb, C. et al, (1980 J. Nucl. Med. 21(11): 1043-50; Visser, F. C. et al, (1985) Circulation 72(3): 565-71). The clinical utility of radiolabeled iodoalkyl FAs appears limited by: 1) the rapid appearance of free radioiodine, requiring special correction procedures to differentiate between myocardial and blood pool activity; 2) short elimination half-lives, making them unattractive agents for single photon imaging; and 3) data suggesting that the elimination rate may not reflect β-oxidation but rather de-iodination and back-diffusion of the tracer across the membrane. Further, protocols and algorithms developed for planar imaging are not applicable to single photon imaging (SPECT), effectively eliminating it as a potential imaging modality for the measurement of metabolic parameters with these radiotracers. Imaging difficulties associated with de-iodination of IHDA and IHXA ultimately resulted in the development of the branched FAs.
The molecular structure of FAs can be modified to attenuate myocardial metabolism, prolong cardiac retention, and avoid washout effects. To prevent rapid de-iodination of alkyl FAs and promote stabilization of the iodine radiolabel, 15-(p-iodophenyl)pentadecanoic acid (IPPA) was developed as an alternative (Machulla, H. et al (1980) Eur. J. Nucl. Med. 5: 171-173). The 123I-label attached to a terminal phenyl ring in either the ortho or para position is stabilized against de-iodination. [123I]-IPPA has kinetics similar to the physiological substrate 11C-PA in perfused rat hearts (Reske, S. et al, (1984) J. Nucl. Med. 25: 1335-1342). The uptake of IPPA is related to perfusion, and IPPA generally follows the normal metabolic pathway for β-oxidation (Caldwell, J. et al, (1990) J. Nucl. Med. 31: 99-105). Iodobenzoic acid and its metabolite iodohippurate are the products of IPPA oxidation, which are rapidly excreted by the kidneys with the iodine moiety still attached, preventing buildup of free radioiodine.
Studies using myocardial biopsy specimens have shown rapid extraction by normal myocardium, with biexponential clearance, including a fast component t1/2 of 3.5 minutes (flow), a slow component t1/2=130 minutes (metabolism), and a blood clearance t1/2=5 minutes (elimination) (Chien, K. et al (1983) Am. J. Physiol. 245: H693-H697). Compared with alkyl straight-chain FAs, IPPA has the advantages of rapid myocardial uptake, iodine stabilization, and rapid clearance of metabolites from the body. While IPPA was a significant improvement over the straight chain FAs, providing excellent image quality, and permitting SPECT image acquisition and quantification with estimates of metabolic rates, the rate of IPPA metabolism and clearance was still relatively fast for SPECT imaging. Thus, an effort was made to develop radiolabeled FA analogs with attenuated oxidative metabolism.
Methyl branching was introduced to slow myocardial clearance and improve quantitative image accuracy (Livni, E. et al. 1982 J. Nucl Med. 23: 169-75; Elmaleh, D. R. et al. 1981. J. Nucl. Med. 22: 994-9; Elmaleh, D. R. et al. 1983 Int. J. Nucl. Med. Biol. 10: 181-7; Goodman, M. M. et al. 1984 J. Org. Chem. 49: 2322-5; Livni, E. et al. 1985. Eur. Heart J. 6 (Suppl B): 85-9; Bianco, J. A. et al. Eur. J. Nucl. Med. 12: 120-4). The addition of methyl group(s) at the 3-carbon position blocks β-oxidation by preventing formation of the 3-carboxy-intermediate (β-ketoacyl-ScoA) via the dehydrogenation of the 5-L-hydroxy ScoA intermediate. Two iodine-labeled MFAs that provide prolonged myocardial retention are 15-(p-iodophenyl)-3-(R,S)-methyl-pentadecanoic acid (BMIPP) and 15-(p-iodophenyl)-3,3-dimethyl-pentadecanoic acid (DMIPP). The kinetics and subcellular distribution of these methyl-branched FAs have been evaluated, with DMIPP demonstrating the greatest myocardial retention times, with no significant metabolism (Knapp, F. et al, (1986) Eur. J. Nucl. Med. 12: S39-S44; Ambrose, K. et al, (1987) Eur. J. Nucl. Med. 12: 486-491). However, DMIPP demonstrates very slow myocardial clearance (6-7 hours), limiting its usefulness for certain applications. Additionally, DMIPP has been detected in exogenous tissues, such as the liver (Sloof et al, (1997) Nucl. Med. Commun. 18(11): 1065-70). BMIPP also accumulates in the liver, but to a lesser extent than BMIPP.
BMIPP is currently the most widely used radiolabeled MFA for cardiac imaging. BMIPP has prolonged myocardial retention (30-45 minutes) and undergoes β-oxidation in the myocyte after the initial α-oxidation and oxidative decarboxylation, producing α-hydroxy-BMIPP as an intermediate (Yamamichi, Y. et al, (1995) J. Nucl. Med. 36: 1042-1050). After loss of propionic acid, further degradation proceeds through successive cycles of β-oxidation to the end product, (p-iodophenyl)-acetic acid. Additionally, it has been shown that initial distribution of BMIPP in the first several minutes after injection is comparable to that of perfusion tracers like 201Thallium and 99mtechnetium compounds. Thus, it can be argued that the use of BMIPP alone imaged early and late after injection is all that is required to evaluate myocardial viability with a high degree of accuracy. Likewise, numerous groups have accomplished work on FAs as potential markers for blood flow and their collective data determined that a single injection of certain FAs produces images that are similar to those produced by 201Tl, 11C-PA, or potassium (van der Wall, E. E. et al, (1980) Eur. J. Nucl. Med. 5(5): 401-5; Kairento, A. L. et al, (1988) Int. J. Rad. Appl. Instrum. B. 15(3): 333-8; Kobayashi, H. et al, (1997) J. Nucl Med. 39(7): 1117-22; Kawamoto, M. et al, (1994) J. Nucl. Cardiol 1(6): 522-8). Thus, a single injection limits the amount of radioactivity exposure to the patient, as well as being cost-effective. While BMIPP is a widely used FA for both flow and metabolism, there is a need in the art for MFAs that have longer myocardial retention without appreciable metabolism and migration of the radiolabel to other unwanted areas. BMIPP and its metabolites have been detected at significant levels in lung tissue (Sloof et al, (1997) Nucl. Med. Commun. 18(11): 1065-70).
While there are a plurality of MFAs that are suitable for imaging, many either undergo significant β-oxidation, resulting in production of radiolabeled metabolites that can accumulate in other tissues, or fail to be efficiently transported into the myocardium, resulting in back-diffusion and re-esterification into the triglyceride pool. Thus, there is a need in the art for a radiolabeled MFA analog that is transported to the tissue of interest by endogenous, physiological means, but fails to undergo β-oxidation due to the presence of branched organic substituents. This would allow the MFA to be retained in a virtually unmodified form for a sufficient amount of time in the tissue of interest (i.e. cardiac tissue) to be detected by conventional means. Further, there is a need in the art for a radiolabeled MFA that shares the aforementioned characteristics and is retained without significant migration to unwanted areas of the organism.